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Figure S1. Alignment of protein sequences of all ALC orthologues and related proteins of full length (or close to full length) identified from databases. The alignment was made using ClustalW through BioEdit (www.mbio.ncsu.edu/BioEdit/bioedit.html), with manual adjustments. Amino acids that are conserved in ≥40% of all sequences are blocked, with amino acids with similar chemical properties shown in the same colour. Sequences with similarity to ALC were identified using the 62 amino acid regions comprising the bHLH domain in addition to 4 N-terminal residues (one part of the bipartite nuclear localisation sequence (NLS)) and 9 C-terminal amino acids (a conserved beta-strand similar to that in SPT – see Groszmann et al., 2008) of AtALC. Sequences are arranged below AtALC by progressively increasing taxonomic distance for ALC, then SPT, and finally two A. thaliana sequences of related protein AtPIF3 and distantly related AtHEC1. Within the 62 amino acid extended bHLH region, residues 3,5,7,8,29,59, and 61 are key residues differentiating ALC orthologs from SPT orthologs. Species in which ALC orthologs were identified are all from within the Brassicaceae, while SPT orthologs were identified from a wide variety of angiosperms. ALC contains a predicted amphipathic helix near the N-terminus similar to the functionally significant amphipathic helix identified in SPT (Groszmann et al., 2008). ALC orthologs lack the conserved, functionally important acidic domain found in SPT orthologs (Groszmann et al., 2008), although there is some sequence conservation in this region.

Sequences are from:

AtALC - Arabidopsis thaliana - At5g67110

AlALC - Arabidopsis lyrata - Derived from whole genome sequencing data GenBank EFH43224

BolC.ALC.b - Brassica oleracea - Derived from whole genome shotgun library GenBank BZ068174

BolC.ALC.a - Brassica oleracea - GenBank BH586940 - Described by Hua et al., 2009

BnaC.ALC.a - Brassica napus - GenBank EV007981- Described by Hua et al., 2009

BnaA.ALC.a - Brassica napus - Described by Hua et al., 2009

BraA.ALC.a - Brassica rapa - Derived from whole genome sequencing data – Brassica rapa database (BRAD) gene 012133

RsALC - Raphanus sativus - Derived from an EST sequence GenBank EX904678

AtSPT - Arabidopsis thaliana - At4g36930

AlSPT - Arabidopsis lyrata - Derived from whole genome sequencing data GenBank EFH43224

BraA.SPT.a - Brassica rapa - Derived from sequenced BAC clone GenBank AC232512

BnaX.SPT.a - Brassica napus - Partially derived from EST TC92993 and remaining sequence derived from EST GenBank DY020422

BraA.SPT.b - Brassica rapa - Derived from partially sequenced BAC clone GenBank CU695342

SlSPT - Solanum lycopersicum (tomato) - TC174571- Described in Groszmann et al., 2008

VvSPT - Vitis vinifera (grape) - Derived from whole genomic shotgun library GenBank AM434294 - Described in Groszmann et al., 2008

MtSPT - Medicago truncatula - Derived from TC97958 and from genomic clone GenBank AC144431 - Described in Groszmann et al., 2008

PpSPT - Prunus persica (peach) - Derived from EST clone TC3088 – Subsequently described in Tani et al., 2011

FaSPT - Fragaria X ananassa (strawberry) - Derived from GenBank AY679615 - Described in Groszmann et al., 2008

GmSPT - Glycine max (soybean) - Soybean TC207452 and from genomic clone GenBank AC170860 - Described in Groszmann et al., 2008

EcSPT - Eschscholzia californica (California poppy) - Described in Groszmann et al., 2008

GhSPT - Gossypium hirsutum (cotton) - Derived from cDNA sequences, GenBank DT572940 and DN780013 - Described in Groszmann et al., 2008

CrSPT - Catharanthus roseus (Periwinkle) - Derived from cDNA clone GenBank ACM41588

RcSPT.a - Ricinus communis (Castor bean) - Derived from whole genomic sequencing database GenBank EEF38808

CcSPT - Citrus clementina (orange) - Derived from cDNA sequence GenBank DY289192 - Described in Groszmann et al., 2008

CpSPT - Carica papaya (Papaya) – Derived from whole genomic sequencing database GenBank CP0021G0176

PtSPT - Populus trichocarpa (poplar) JGI eugene3.00400325 – Described in Groszmann et al., 2008

RcSPT.b - Ricinus communis (Castor bean) - Derived from whole genomic sequencing database GenBank EEF52377

AtPIF3 - Arabidopsis thaliana - AT1G09530

AtHEC3 - Arabidopsis thaliana - AT5G09750

Protein sequences related to AtALC were obtained using BLAST searches against all available datasets from the following databases:

JGI Arabidopsis lyrata genomic sequence database v1.0 (http://genome.jgi-psf.org/Araly1)

Brassica Database - BRAD sequences (http://brassicadb.org/brad/)

Brassica gateway dataset (http://brassica.bbsrc.ac.uk/)

NCIB (http://blast.ncbi.nlm.nih.gov/)

TIGR gene indices - TC sequences (http://compbio.dfci.harvard.edu/tgi/plant.html)

Plant Genomic Databases (http://www.plantgdb.org/CpGDB/) [includes the Caricaceae (papaya) genome±]

Cleome EST datasets (Barker et al., 2009*) (limited – could not find ALC nor SPT within this limited EST dataset derived from leaf tissue)

Not all identified SPT orthologs (Groszmann et al., 2008) have been included in this alignment.

Hua, S.J., Shamsi, I.H., Guo, Y., Pak, H., Chen, M.X., Shi, C.G., Meng, H.B. and Jiang, L.X. (2009) Sequence, expression divergence, and complementation of homologous ALCATRAZ loci in Brassica napus. Planta, 230, 493-503.

±Ming, R., Hou, S.B., Feng, Y. et al. (2008) The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). Nature, 452, 991-997.

*Barker, M.S., Vogel, H. and Schranz, M.E. (2009) Paleopolyploidy in the Brassicales: Analyses of the Cleome transcriptome elucidate the history of genome duplications in Arabidopsis and other Brassicales. Genome Biology and Evolution, 1, 391-399.

Figure S2. Phylogeny of identified SPT and ALC orthologues across the angiosperms. Sequences were from this study (Figure S1) and Groszmann et al., (2008) showing that no ALC orthologues have been identifed outside the Brassicales. The phylogeny was created using whole protein sequences utilising the Neighbour-Joining algorithm with pairwise deletions and a bootstrap of 10,000 replications. Bootstrap valves > 50 are in red. Note that the separation between the ALC and the SPT clades shows a high supporting value.

Figure S3. Angiosperms in which SPT and ALC orthologues have been identified. Phylogeny of Angiosperm orders with taxonomic placements based on APG II, 2003 (http://www.mobot.org/mobot/research/apweb). Blue arrows indicate that a SPT orthologue(s) has been identified within a species contained in that order (present study; Groszmann et al., 2008). Red arrow indicates that both SPT and ALC orthologues have been identified within species contained in that order. The value in parentheses indicates the number of orthologues identified in that order.

Figure S4. Defects in spt-2 alc-1 double mutant plants. (a) Frequency of development of stigmatic papillae in spt-2 versus spt-2 alc-1 double mutant flowers (all wild type flowers develop stigmatic papillae). Five representative spt and spt alc plants were selected and scored for stigmatic differentiation in the first 20 gynoecia produced on the main stem. Although stigmatic papillae did eventually differentiate in the spt alc mutant their abundance and length were reduced compared with that observed for spt single mutants. (b) SEMs of the basal regions of wild-type (WT) and spt alc double mutant gynoecia at stage 13. WT gynoecia have clearly defined valve margins (arrows) easily identified by the constrained growth between valve and replum. spt alc double mutant gynoecia lack clearly defined valve margins (arrows), especially at the extreme apical regions (see Figure 3l) and basal regions of the valves (yellow arrows). (c) spt alc double mutants show defects in septum development. Transverse sections of developing spt alc gynoecia showing hindered periclinal divisions in the medial ridge [mr] from a stage 8-9 gynoecium, leading to a reduction in early septum [s] development during stages 9-10, and subsequently leading to the two septal halves failing to unite and produce a central pool of transmitting tract tissue during stage 12. (For wild type and spt-2 mutant controls, see Alvarez and Smyth, 2002.)

Figure S5. Tissue specific expression of SPT and ALC during different stages of Arabidopsis development. Assays of tissue specific expression of SPT and ALC obtained from publicly available micro-array experiments (Schmid et al., 2005). Note that ALC is expressed far more strongly from stage 9 onwards in all samples incorporating the gynoecium, coinciding with the strong VM and valve expression observed using the ALC:GUS reporter.

Figure S6. Nucleotide sequence alignment comparing the AtALC upstream sequence against the upstream sequences of five ALC orthologues.

Identical nucleotides are blocked. A consensus sequence (Cons) was generated. Seven regions were identified as containing significant homology between AtALC and the other sequences (coloured blocks). Highlighted in red are conserved TATA-box sequences and the AtALC start of transcription (+1) as determined from the longest ALC cDNA available: Genbank (AY084632).

Upstream sequences are from:

AtALC – Arabidopsis thaliana. Derived from TAIR9 database

AlALC – Arabidopsis lyrata. Derived from whole genome sequencing data - JGI scaffold 8: 22454884-22455831

BraA.ALC.a - Brassica rapa - Derived from whole genome sequencing data – Brassica rapa database (BRAD) gene 012133.

BolC.ALC.a – Brassica oleracea. Derived from whole genomic shotgun library GenBank BOGKS90TR.

BolC.ALC.x (very similar to BolC.ALC.a – but insufficient overlap to accurately determine) – Derived from whole shotgun library GenBank BZ011605.

BolC.ALC.c – Derived from whole genomic shotgun library GenBank BZ027462.

Figure S7. Combined mutants of spatula and fruitful, and of spatula and shatterproof1/2. (a-c) spt-2 ful-5 double mutants. (a) Inflorescence of a spt-2 ful-5 double mutant plant. Note the reduction in gynoecium growth, especially at dehiscence stage 13 flowers, to the point where anthers dehisce well above the under-developed gynoecium (arrow). (b) A whole mount medial view of a typical stage 11 spt-2 ful-5 flower that has an aberrantly developed style more severely disrupted than seen in the spt-2 single mutant. The spt-2 ful-5 style comprises of two unfused regions of stylar tissue with tufts of stigmatic papillae capping each. The two stylar growths are longer than the style from an equivalent aged WT gynoecium (see Figure 3e). (c) Comparison between gynoecia of a spt-2 ful-5 double mutant (left) and a spt-2 single mutant (right), both at stage 13. The spt-2 ful-5 double mutant has a more elongated and unfused stylar region. (d) spt-2 shp1 shp2 triple mutant siliques. There is no apparent enhancement of the disruption to fusion of the style or upper ovary normally associated with the spt-2 single mutant at stage 13 (left image, compare (c)). At maturity (stage 17) the spt-2 shp1shp2 triple mutant silique shows an additive affect producing a spatula-shaped indehiscent silique (medial view middle image; lateral view right image). Bar = 50 μm.

Table S1. Overlap between SPT and ALC expression patterns. Presence (+) or absence (-) of SPT and ALC expression in a range of tissues identifying regions of co-expression. Summary of ALC expression is from this study. Summary of SPT expression is from Groszmann et al. (2010). Expression patterns within developing gynoecium and silique tissues are very similar except that ALC and SPT are expressed differentially: (1) in the valves, with ALC expressed alone between stages 9-11, with both SPT and ALC expressed during stages 12-13 but ALC only in the epidermis and SPT throughout, and with SPT only from stage 14; (2) in the septum where ALC expresses in the epidermis only, whereas SPT expresses throughout; and (3) in the ovules where ALC expression is limited to the embryo sac, whereas SPT is found in other ovule tissues, including funiculi.

Table S2. Co-expression in onion epidermal cells shows that SPT can dimerise with itself and with ALC. Dimerisation was assayed based on the number of fluorescent cells recorded showing nuclear localised fluorescence of the 35S:SPT(ΔNLS)-GFP construct co-inserted with either 35S:SPT, or 35S:ALC, using a scale of; ‘+++++’ very strong to ‘+’ very weak and ‘-’ no fluorescence. A control involving the NLS deleted construct alone showed both nuclear and cytoplasmic fluorescence.

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